I. Field of the Invention
The present invention relates to the fields of oncology, genetics and molecular biology. More particular the invention relates to the use of two probes for regions of human chromosomes 3 and 10 that are highly predictive of the development of neoplasia and progression of neoplastic events.
II. Related Art
Lung cancer is one of the leading causes of cancer death in the world. The high mortality rate for lung cancer probably results, at least in part, from the lack of standard clinical procedures for the diagnosis of the disease at early and more treatable stages compared to breast, prostate, and colon cancers. There is also extremely poor prognosis associated with diagnosis of the disease, especially in advanced disease. It is important that strategies to detect early stage lung carcinoma or its precursors, such as atypical squamous metaplasia, dysphasia and carcinoma-in-situ in subjects at high risk be devised.
Cigarette smoking over a prolonged period of time is the most important risk factor in the development of lung and other smoking related cancers, with other risk factors including exposure to passive smoking, certain industrial substances such as arsenic, some organic chemicals, radon and asbestosis, ingestion of alcohol, radiation exposure from occupational, medical and environmental sources, air pollution and tuberculosis. Many of these factors greatly increase the risk of development of lung and other smoking related cancers if they occur in a person who is concurrently a smoker.
Genetic detection of human disease states is a rapidly developing field (Taparowsky et al., 1982; Slamon et al., 1989; Sidransky et al., 1992; Miki et al., 1994; Dong et al., 1995; Morahan et al., 1996; Lifton, 1996; Barinaga, 1996). However, some problems exist with this approach. A number of known genetic lesions merely predispose to development of specific disease states. Individuals carrying the genetic lesion may not develop the disease state, while other individuals may develop the disease state without possessing a particular genetic lesion. In human cancers, genetic defects may potentially occur in a large number of known tumor suppresser genes and proto-oncogenes.
The genetic detection of cancer has a long history. One of the earliest genetic lesions shown to predispose to cancer was transforming point mutations in the ras oncogenes (Taparowsky et al., 1982). Deletion and mutation of p53 has been observed in bladder cancer (Sidransky et al, 1991). Numerous studies have shown deletions in the 3p region are related to lung and other smoking related cancers (Mitsudomi et al., 1996, Shiseki et al., 1996, Wistuba et al., 2000, Wu et al., 1998, and Shriver et al., 1998).
Molecular studies (fluorescence in situ hybridization (FISH) for polysomies, PCR for hypervariable markers (MI) and LOH, or specific mutations) have demonstrated that morphologically normal areas of bronchial epithelium closest to the carcinomas frequently show the most molecular abnormalities (3p, 17p, 9p, 5q). In particular, the short arm of chromosome 3 has been shown to frequently harbor deletions of alleles in several regions including 3p25-26, 3p21.3-22, 3p14 and 3p12. These regions are presumed to be the site of tumor suppressor genes, and loss of chromosome 3p allelles have shown to be an early event in lung tumorigenesis.
Chromosomal alterations in several cancers have been investigated, and frequent LOH at chromosome 10 has been reported in a variety of cancers, including glioma, glioblastoma multiforme, prostate cancer, endometrial cancer, chondrosarcome, bladder cancer, malignant melanoma, and follicular thyroid tumors ((Licciardello et al., 1989; Auerbach et al., N. Engl J. Med., 265: 253-267, 1961; Voravud, et al., 1993; Feder et al., 1998; Yanagisawa et al., 1996; Thiberville et al., 1995; Papadimitrakopoulou et al., 1996; Zou et al., 1998; Brugal et al., 1984; Dalqueni et al., 1997; Muguerza et al., 1997).
Deletion rates of chromosome 3p are known to correlate with lung cancer. However, there is no current clinical method for the identifying a population of individuals who are at a high risk to develop lung cancers or upper airway primary or secondary cancers. A technique for determining the risks of developing these cancers would be of great value for the ability to limit exposure to additional environmental risk factors and to know when additional tests, supplements, or treatments are appropriate.
In various studies, chromosome deletions have been studied as identifiers for lung cancers. For example, Shiseki et al, (1996) analyzed 85 loci on all 22 autosomal chromosomes to determine that the incidence of LOH on chromosome arms 2q, 9p, 18q, and 22 q in brain metastases were significantly higher than that in stages I primary lung tumors. Mitsudomi et al. (1996) used PCR-based analysis for the detection of LOH in non-small cell lung cancer. Multiple regions on chromosome 3p were observed to show that deletions of the 3p chromosome may help to identify non-small cell lung cancer patients with a poor prognosis. Wistuba et al. (2000) used fifty-four polymorphic markers used to study the entire chromosome arm 3p and concluded that 3p allele loss is nearly universal in lung cancer pathogenesis. Wu et al. (1998) studied 3p21.3 deletion using the probe, D3S4604/luca. Peripheral blood lymphocytes of 40 lung cancer patients were observed to give the conclusion that lung cancer patients exposed to benzo[xcex1]pyrene, a common byproduct of tobacco smoke, have frequent deletions in peripheral blood lymphocytes. Shriver et al. (1998) studied lung cancer cell lines and identified the human homolog of the L14 ribosomal protein gene, RPL14; deletion of RPL14 was shown to be related to the development of lung cancer. None of theses studies, however, are able to predict the susceptibility of a patient to the development of lung cancer or to predict whether smokers and non smokers are at a high risk of developing lung or other smoking related cancers.
Because of the grim prognosis of lung cancer with a ten year survival rate of  less than 5% the only curable cancers are those diagnosed in the early stages and treated surgically. There is a shift of interest towards diagnosis and study of early and preneoplastic states. Because early detection and effective chemoprevention therapy have potential to be curative, it is imperative to stratify the patients in clinical trials. These patients need to be monitored fore results of chemoprevention therapy and also for predictions whether a particular preneoplastic lesion may progress.
The present invention provides probes located on chromosomes 3p21.3 and 10q22 useful in the diagnosis and prognosis of cancers related to smoking. In one embodiment, a method for identifying a subject at high risk for the development, recurrence, or metastasis of cancer comprising the steps of (a) obtaining a test sample from a subject; (b) providing a nucleic acid probe targeting RPL14, CD39L3, PMGM, or GC20; (c) contacting the probe with the test sample; and (d) analyzing DNA from the sample whereby aberrations in the hybridization of said probe to said DNA was compared to wild type DNA, indicating the risk for the development, recurrence, or metastasis of cancers.
More specifically the method identifies the risk for the development of cancers. The cancer may be lung, upper airway primary or secondary, head or neck, bladder, kidneys, pancreas, mouth, throat, pharynx, larynx, esophagus, brain, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, bone marrow and blood cancer. In preferred embodiments, the cancer is lung cancer. The test sample can include, but is not limited to, a surgical or biopsy specimen, paraffin embedded tissue, frozen tissue, surgical fine needle aspirations, bronchial brushes, bronchial washes, bronchial lavages, buccal smears, sputa, peripheral blood lymphocytes, esophageal brush, a fine needle aspiration, urinary specimens such as bladder washings and voided urine, and esophageal washes.
In one embodiment, it is provided that the subject can come from a group comprising smokers, former smokers, or non-smokers. In a similar embodiment, the test sample comes from said subject who has not previously been diagnosed with cancer.
It is a further embodiment of this invention that additional testing, agents or treatments may be performed after the risk for the development of said cancers has been analyzed. This includes, but is not limited to, a spiral CT-scan, cancer therapies and pharmaceutical treatments which can include radiotherapeutic agents, surgical treatment for removal of the cancerous growth, chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, biological modifying respidase drugs and other agents. These agents and treatments can be used alone or in combination with other agents.
In certain embodiments, it is contemplated that FISH is used to measure the aberrations in the particular loci. A unique 3p21.3 probe can be from 1000 to 2000 base pairs or larger and used for detection in a region of about 180,000 base pairs. The probe can be labeled with a fluorophore, or more specifically digoxigenin. A specific 10q22 probe can be used in conjunction with the 3p21 probe. In certain embodiments, a control probe is used which can be labeled with a fluorophore, or more specifically spectrum orange. The control probe is a chromosome 3 stable marker or more specifically Centromere 3 (CEP 3).
In another embodiment, there is provided a method for identifying a subject at high risk for the development, recurrence, or metastasis of cancer comprising: (a) obtaining a lung test sample from a subject; (b) providing a specific10q22 DNA probe; (c) contacting said probe with said test sample; and (d) analyzing DNA from said test sample, whereby aberrations in the hybridization of said probe to said DNA is compared to wild type DNA, indicating the risk for the development, recurrence or metastasis of said cancers. More specifically the method identifies the risk of the recurrence or metastasis of cancers. In a further embodiment, the probe size is from 1000 to 2000 base pairs or larger, for detection in a region of about 200,000 base pairs. In an additional embodiment, a specific 3p21 probe can be used with the 10q22 DNA probe. The control probe is a chromosome 10 stable marker, or more specifically Centromere 10 (CEP10).
In another embodiment, there is provided a method for predicting the progression or metastasis of non-small cell carcinoma and other carcinoma in a subject comprising: (a) obtaining a test sample from a subject; (b) providing a RPL14, CD39L3, PMGM, or GC20 gene probe; (c) contacting said probe with said test sample; and (d) analyzing DNA from said test sample.
In yet another embodiment, there is provided a method for predicting the progression or metastasis of non-small cell carcinoma in a subject comprising: (a) obtaining a lung test sample from a subject; (b) providing a specific10q22 DNA probe; (c) contacting said probe with said test sample; and (d) analyzing DNA from said test sample.
In a further embodiment, there is provided a method for the staging lung of cancer in a subject comprising determining the deletion distribution of the 3p21.3 region.
In one embodiment, there is provided a method of determining likelihood of relapse or a new primary for a cancer subject comprising determining genetic aberrations at chromosomal loci 3p21.3 or 10q22 in DNA of bronchial tissue adjacent to tumor tissue from said subject, wherein abnormalities in DNA of said adjacent tissue correlate with relapse of said cancer. The cancer can comprise lung cancer or more specifically non-small cell carcinoma, adenocarcinoma, or squamous cell carcinoma. A specific gene probe may comprise RPL 14, CD39L3, PMGM, or GC20, or a 10q22 DNA probe. The 10q22 probe lies adjacent to the PTEN gene which is frequently involved non-small cell cancer. Both the 3p and the 10q probe can be used simultaneously. The test sample can be chosen from the same or contralateral lung, and can consist of tumorous or nontumorous bronchial cells.
In yet another embodiment, there is provided a method of identifying an individual to be segregated from a high risk environment comprising: (a) obtaining a test sample from a subject; (b) providing a gene probe containing RPL14, CD39L3, PMGM, and GC20 genes and PTEN or a 10q22 DNA probe, (c) contacting said probe with said test sample; and (d) analyzing DNA from said test sample, whereby said analysis is used to identify an individual who is highly susceptible to the development of lung cancer and who should not be exposed to a high risk environment.